Ovarian cancer may be a prototypical stem cell disease since of its 25,000 new cases per year in the United States 70% recur and die despite early favorable initial clinical responses to surgery and cytotoxic drug therapy. Little progress has been made in improving cure rates as early peritoneal seeding and metastatic spread accounts for the fact that less than 25% of women are diagnosed at the more favorable stage I. The critical barrier to improvement in ovarian cancer therapy is the high incidence of recurrence often attributed to multi-drug resistance; thus, novel therapies must be found. Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics.
Mullerian Inhibiting Substance (MIS) (Teixeira et al. 2001), a 140 kDa glycoprotein disulfide linked homodimer secreted by fetal testes, causes Mullerian duct regression in vertebrate embryos. Epithelial ovarian cancer recapitulates the original histology of the embryonic Mullerian ducts and its various subtypes (Scully 1977); for example, serous cystadenocarcinoma resembles embryonic Fallopian tube, endometrioid carcinoma, the endometrium, and mucinous carcinoma, the cervix. MISRII is expressed in the majority of epithelial ovarian cancers (Masiakos et al. 1999; Bakkum-Gamez et al. 2008; Song et al. 2009) and MIS inhibits their growth in vitro and in vivo, without obvious toxicity after prolonged therapy in vivo (Pieretti-Vanmarcke et al. 2006b). Also, MIS acts synergistically or additively with commonly used cancer drugs to control tumor growth (Pieretti-Vanmarcke et al. 2006a). There is a growing body of research reporting that ovarian cancers and cell lines are heterogeneous, with populations that are resistant to drugs but remain responsive to MIS. MIS particularly targets a population (3+) of cells with stem/progenitor characteristics that respond poorly to chemotherapeutic agents currently in clinical use for ovarian cancer (Wei et al, 2010).
Isolation and molecular scrutiny of a stem/progenitor-enriched population of cancer cells may provide new insights into events leading to early transformation or initiation, progression, maintenance, and recurrence of human ovarian cancers. The identification of markers for these cancer stem/progenitor cells in human ovarian cancer is important in order to develop markers for earlier detection, to discover new therapeutic targets, and to modify therapeutic protocols to include stem cell targets.
The inventors have previously isolated cells by exclusion of Hoechst 34422 dye enriched for a “side population”, where these cells had stem/progenitor properties in ovarian cancers from transgenic mice and human ovarian cancer cell lines; They have further enriched this unique population by selecting for cells that expressed three markers (CD44, CD24, and Epcam) from a total of 130 markers compatible for use with flow cytometry and thereby isolated a population of more highly purified human ovarian cancer cells endowed with “stem/progenitor” characteristics (Wei et al, 2010). Cells with the CD44, CD24, and Epcam triple positive (3+) markers were detected in primary ovarian cancers, ovarian cancer cell lines, and normal fimbria as a surrogate cell of origin of ovarian epithelial carcinomas (16), based on the hypothesis that normal somatic and tumor stem/progenitor cells would share many molecular characteristics. The 3+ cells showed stem cell characteristics of 1) shorter tumor free intervals after limiting dilution in vivo and 2) increased colony formation and 3) enhanced migration in vitro.
Stem cells are thought to have properties of pluripotency, defined as the ability to differentiate into multiple cell lineages. Recent studies have shown that it is possible to reprogram adult somatic cells by overexpression of key pluripotency factors. Four transcription factors, Oct3/4, Sox2, KLF4, and c-Myc, were found to be sufficient to induce pluripotency in adult mouse (Takahashi, 2006) and human (Park, 2008) fibroblasts. Subsequently, a substitution of the microRNA-binding protein, Lin28, for c-Myc also was successful in reprogramming human fibroblasts (Yu, 2007; Lako, 2009). Since Lin28 overexpression has been detected in a number of human cancers, including ovarian cancer (Viswanathan, 2009), and has been shown to derepress Let-7, a known microRNA capable of suppressing oncogenes, we studied Lin28 expression in ovarian cancer cell lines and transgenic cancers. Lin28, through its miRNA target Let-7, is thought to regulate the expression of cell cycle-related genes and to contribute to cancer stem/progenitor cell self-renewal and differentiation.